A major hurdle facing the promising therapy of islet transplantation for patients of type I diabetes is the shortage of cadaver pancreatic islets. A potential solution to this problem is the generation of insulin-producing, glucose-responsive beta cells from embryonic stem (ES) cells. Several groups have shown progress in differentiating mouse ES cells into insulin-producing cells, however, the amount of insulin output has been very low. In our laboratory, we have established a differentiation protocol that results in cells that express insulin levels higher than previously published. The goal of this proposal is to increase the output of insulin-expressing cells from our culture system through genetic manipulation of beta cell progenitors using neurogenin 3 (ngn3), which is a transcription factor necessary for pancreatic endocrine development. Previous studies suggest that the timing of ngn3 overexpression is critical in driving beta cell generation in embryogenesis. We hypothesize that the forced expression of ngn3 at restricted stages of ES cell differentiation will lead to an increase in the numbers of insulin-expressing cells derived from our culture system. Our first specific aim entails using a doxycycline-inducible ES cell line to express ngn3 at specific time points in order to increase the amount of insulin output from our differentiation system. We will test these insulin-expressing cells for the presence of endodermal and pancreatic markers and for increased levels of insulin. In our second specific aim, we will assess the insulin-expressing cells, derived from the ngn3 ectopic expression, for glucose responsive and in vivo function through transplantation into diabetic mice. Completion of this proposal will lead to a greater understanding of the role of ngn3 in beta cell commitment and progress towards the manipulation of ES cells for therapies to cure diabetes. Relevance: In the past 30 years, transplantation of beta cell-containing islets has evolved into a promising long-term treatment for Type I diabetes. Manipulating embryonic stem cells to generate insulin-producing cells will circumvent the current problem of limited numbers of cadaver islet cells available for transplantation. The research proposed here will contribute to understanding what factors are necessary for establishing large pools of these transplantable, insulin-producing cells. [unreadable] [unreadable] [unreadable]